VL depend on Rac1, cortactin, and Arp2/3. (a) MVECs were coexpressing mDsRed and GFP dominant-negative Rac1 (DN Rac1) or Rho A (DN RhoA) and imaged during T cell diapedesis. VL velocity (i), wound closure time (ii,) and diameter (iii) were measured. (b) MVECs expressing mYFP were imaged during T cell diapedesis and addition of Rac1 inhibitor NSC23766 or vehicle (red phase) and after drug washout (blue phase). The cumulative percentage of wound closure was plotted as a function of time. n = 5. (c) A representative imaging of reversible VL blockade. (top left) Two DIC and mYFP panels show three transcellular pores recently vacated by T cells (T1–T3). Subsequent panels are after addition (red outline) or washout (blue outline) of NSC23766. Arrowhead indicates pore closing VL rapidly mobilized after washout. See Video 9. (d) MVECs coexpressing mDsRed and GFP-fused actin, cortactin, IQGAP, tubulin, Caveolin-1, CD63, Lamp1, VAMP2, and an endosomal marker (ENDO) were imaged during T cell diapedesis. mDsRed-normalized intensities of each GFP construct were quantified in VL versus non-VL regions. n > 5. (e) Representative images of cortactin enrichment both in initiation nodes (N) and VL leading edges (VL; arrowheads). (f) VL velocity (i) and diapedesis wound closure time (ii) in MVECs expressing wild type (WT) or dominant-negative cortactin-GFP (DN). All values are mean ± SEM. Statistical significance is indicated with p-values as follows: ***, P < 0.001; *, P < 0.05. (g, i) Actin-GFP–expressing MVECs were mechanically micro-wounded. VL (arrowheads) that formed specifically in zones of recoil (blue shading) and collapsed upon addition of NSC23766. Green line traces location used for kymograph of this process (ii). (h) Kymograph of MVECs micro-wounded in the presence of Arp2/3 inhibitor CK-666, and VL (cyan arrowhead) initiation after drug washout. n = 10. Bars, 5 µm.